Citations

7041 Background: Prognostic factors for diffuse large B-cell lymphoma (DLBCL) at day 100 post-autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT) have not been evaluated. We previously reported that absolute monocyte (AMC)/lymphocyte count (ALC) prognostic score (AMLCPS) at diagnosis of DLBCL patients is a prognostic factor of survival, stratifying patients into three risk groups: low- (AMC<630/μL and ALC>1000/μL), intermediate- (AMC ≥ 630/μL or ALC ≤ 1000/μL) and high-risk (AMC ≥ 630/μL and ALC ≤ 1000/μL) (Leukemia 25: 1502-9, 2011). Therefore, we evaluated day 100 AMLCPS as a prognostic factor of survival by landmark analysis from day 100 post-APBHSCT in DLBCL patients. Methods: DLBCL patients that achieved complete response by day 100 post-APBHSCT qualified for the study. From 2000 to 2007, 134 consecutive DLBCL patients were evaluated. Overall survival (OS) and progression free survival (PFS) were calculated using Kaplan-Meier analysis. Results: The median follow up from day 100 for the cohort was 5.5 years (range: 0.17-12.17 years) and for living patients (N=93) was 6.9 years (range: 2.5-12.17 years). Day 100 AMLCPS was able to stratify patients into three risk groups low-, intermediate-, and high-risk for OS [low: median OS = not reached, 5-year survival rate = 94% (95%CI 83.3%-98.1%); intermediate: median OS = not reached, 5-year survival rate = 70% (95%CI 58.0%-79.8%); high: median OS = 2.2 years, 5-year survival rate = 13% (95%CI 3.4%-40.5%); p<0.0001] and PFS [low: median PFS = not reached, 5-year progression free rate = 87% (95%CI 74.5%-94.3%); intermediate: median PFS = 10.9 years, 5-year progression free rate = 67% (95%CI 55.4%-77.5%); high: median PFS = 1 year, 5-year progression free rate = 13% (95%CI 3.4%-40.5%); p<0.0001]. The day 100 AMLCPS was balanced in relation to the International Prognostic Index (IPI; p=0.22), infused CD34+ stem cells (p=0.92), and disease status pre-APBHSCT (complete remission versus partial response; p=0.11). Conclusions: The day 100 AMLCPS is a simple biomarker that can help identify DLBCL patients post-APBHSCT with poor clinical outcomes.

3037 Background: Tumor immunotherapies directed towards enhancing natural or endogenous anti-tumor T-cell immunity in patients with advanced malignancies are currently being implemented in clinic with promising results. In order to optimize therapeutic protocols and monitor the effectiveness of such therapies, a reliable T-cell marker is needed. Methods: We utilized CD11a (LFA-1, lymphocyte functional-associated antigen 1), an integrin up-regulated on effector and memory CD8 T-cells, and PD-1 (programmed death-1), an immunoregulatory receptor expressed by activated T cells, as biomarkers to identify, quantitate and monitor endogenous tumor-reactive cytotoxic lymphocytes (CTLs) in two mouse tumor models and the peripheral blood (PB) of 12 patients with stage IV melanoma. Results: High expression of CD11a and PD-1 was identified among CD8 T-cells residing within primary and metastatic murine tumor sites, as well as in spontaneous murine breast cancer tissues. In the PB of melanoma patients, tumor antigen-specific CD8 T cells were associated with a population of CD11a high CD8 T-cells which co-expressed high levels of PD-1, as opposed to eleven healthy donors who had a much lower frequency of PD-1+ CD11a high CD8 T-cells (p <0.0001). Phenotypic analysis showed that CD11a high CD8 T-cells are proliferating (Ki67 positive) activated (CD62L low, CD69 high) T-cells. Increased CD11a high CD8 T-cells and delayed tumor growth were observed in PD-1 deficient mice, suggesting that the antitumor effector function of CD8 T cells is compromised by co-expression of elevated levels of PD-1. Conclusions: CD11a high CD8 T-cell population expresses high levels of PD-1 and is likely the cellular target of PD-1 blockade therapy. High expression of CD11a (LFA-1) and PD-1 (CD279) by CD8 T-cells may represent a novel biomarker to identify and monitor endogenous tumor-reactive CTLs. This may not only provide an immunological readout for evaluating the efficacy of therapy, but also contribute to the selection of patients with solid malignancies likely to benefit from anti-PD-1 therapy.

e21112 Background: Metastatic malignant melanoma is difficult to treat and has a poor prognosis. Systemic therapy for metastatic melanoma has had limited success and novel drug combinations need to be tested. To that end, we have combined anti-angiogenic therapy (bevacizumab) with chemotherapy in clinical trials and have shown some clinical efficacy. Vascular endothelial growth factor (VEGF) is increasingly seen as an immunosuppressive factor that hampers dendritic cell maturation and reduces antigen presentation. We have recently shown that tumor-derived VEGF plays a role in Th-2 driven systemic chronic inflammation seen in advanced melanoma patients. Therefore, our data suggest that blocking tumor derived VEGF with the monoclonal antibody bevacizumab may impact immunity in such a way that augments anti-tumor immunity enhancing clinical efficacy. Methods: In order to understand the immune status in metastatic melanoma patients on chemotherapy (n=55) or chemotherapy plus bevacizumab (n=73), we measured levels of 42 cytokines, chemokines and growth factors in plasma by a mulit-plex bead assay. Matched pairs analysis was performed to determine if the average responses of the measured cytokines were different between the patients that received chemotherapy with bevacizumab or chemotherapy alone. The pre-treatment levels were compared with levels drawn four weeks after the first treatment. Results: There were significant differences in IL-1a, IL-1ra, IL-6, IL-8 and IP-10 between patients treated with chemotherapy alone relative to patients treated with chemotherapy plus bevacizumab. In all cases of patients treated with chemotherapy plus bevacizumab, the levels of these cytokines all decreased relative to pre-treatment samples. In patients treated with chemotherapy alone the levels of these cytokines increased relative to pre-treatment samples. Conclusions: Our data suggest that treating patients with the anti-angiogenic drug bevacizumab may affect the plasma cytokine milieu by reducing the level of many key inflammatory cytokines. Therefore, the use of bevacizumab in combination with chemotherapy may augment host immunity and elicit a more effective anti-tumor immune response.

8600 Background: The prognosis of stage IV melanoma patients remains poor. Published results have suggested that components of the complete blood count have significant prognostic value in several malignancies. Among the most studied were the absolute lymphocyte count (ALC), and absolute monocyte count (AMC) on clinical outcomes of patients with lymphoid malignancies. Thus, we sought to investigate if the pre-operative ALC (ALC-PO), AMC (AMC-PO) and ALC/AMC ratio (ALC/AMC-PO) affects the risk of disease recurrence and survival after complete surgical resection of metastatic melanoma. Methods: We studied 66 stage IV, resected melanoma patients followed at Mayo Clinic from 2000 to 2010. Log rank chi-square analysis was used to determine the best cut-off values for each pre-operative variable, while proportional hazards models were used to compared survival. Results: The median follow-up of the cohort was 24 months (range: 2.3 – 117 months). ALC-PO, AMC-PO and ALC/AMC-PO, as continuous variables, were all identified as prognostic factors for both relapse-free survival (RFS) and overall survival (OS). The best cut-off values for ALC-PO, AMC-PO and ALC/AMC-PO were 1.9; 0.62; and 2.05, respectively. Using Kaplan-Meier analysis, patients with an ALC-PO ≥ 1.9 x 109/L experienced superior OS and RFS compared with ALC-PO < 1.9 x 109/L patients [median OS of 58 months vs. 34 months, p < 0.04; median RFS of 14 months vs. 5 months, p < 0.009]. Conversely, a low AMC-PO (<0.62 x 109/L) was associated with better OS and RFS compared with higher AMC-PO (≥ 0.62 x 109/L): [median OS of 47 months vs. 14 months, p < 0.007; median RFS of 9 months vs. 5 months, p < 0.02]. When the ALC-PO and AMC-PO were combined as an ALC/AMC ratio, the group with an ALC/AMC-PO ≥ 2.05 experienced a superior OS and RFS compared to patients with ALC/AMC-PO < 2.05: [median OS of 49 months vs. 12 months, p < 0.0001; median RFS of 10 months vs. 4 months, p < 0.0001]. Multivariate analysis showed ALC/AMC-PO to be an independent prognostic factor for RFS and OS (HR = 0.32, p < 0.003; HR = 0.23, p < 0.002). Conclusions: Our study showed, that ALC/AMC-PO ratio is an independent prognostic factor for RFS and OS in patients undergoing resection of metastatic (stage IV) melanoma.

e14507 Background: Serious gastrointestinal injury (SGI) is a rare but documented side effect that can occur independently with agents that affect the vascular endothelial growth factor (VEGF) receptor or with stereotactic body radiotherapy (SBRT). The risk of SGI in patients treated with the combination of SBRT sequentially followed by a VEGF inhibitor (VEGF-i) is not yet quantified. We explored the incidence of SGI in patients treated with SBRT with or without VEGF-i therapy at a single institution. Methods: From May 2008 to August 2011, 76 patients with 84 primary or metastatic intraabdominal lesions underwent SBRT (median dose, 50 Gy in 5 fractions). Twenty of these patients (26%) received a VEGF-i within two years of completing SBRT (bevacizumab, n=14; sorafenib, n=4; pazopanib, n=1; sunitinib, n=1). The most common site treated with SBRT was the liver (n=43, 57%), and the most common histology was colorectal adenocarcinoma (n=18, 24%). Other common histologies included melanoma of the skin (n=14, 18%) and non-small cell lung cancer (n=10, 13%). The incidence of SGI (CTCAE v4.0 grade 3 to 5 ulceration or perforation) after SBRT was obtained, and the relationship between SGI and VEGF-i was examined. Other factors that could potentially contribute to SGI, such as radiotherapy dose to bowel, were also analyzed. Results: Seven patients (9%) experienced SGI at a median 4.6 months (range, 3-17 months) after SBRT. All seven had received a VEGF-i prior to SGI and within 13 months of completing SBRT, and five received the VEGF-i within three months of SBRT. The six-month estimate of SGI in patients receiving a VEGF-i within three months of SBRT was 38%. No SGI was noted in the 63 patients not receiving VEGF-i therapy after SBRT. The Log-Rank test showed a significant correlation between SGI and VEGF-i therapy within three months of SBRT (p=0.0006) but not between SGI and maximum radiotherapy bowel dose (p=0.20). Conclusions: The combination of SBRT and VEGF-i therapy results in a much higher risk of SGI than would be expected with either treatment given independently. Local therapies other than SBRT should be considered if a patient is likely to be treated with a VEGF-i in the future.

8599 Background: Chemotherapy resistance is a common, difficult problem for melanoma patients needing long term care and those suffering from recurrence. Neural growth factor receptor (NGFR), also known as CD271, is commonly expressed on mesenchymal stem cells and is important for development, survival and differentiation of cells. This has previously shown to be expressed on melanoma stem cells. We hypothesized that treatment with carboplatin enriches the population of tumor cells for cancer stem cells (CSC) as a mechanism of chemotherapy resistance. Methods: Core tumor samples from 118 patients with metastatic melanoma were obtained from formalin-fixed paraffin embedded specimens. Slides were stained using an anti-CD271 antibody for IHC. A375 melanoma cells were treated with increasing concentrations of carboplatin for approximately 1 year. WT, 6 mg/mL and 10 mg/mL resistant cells were tagged with CD271-PE antibody and run on a flow cytometer. These cells were then tested for gene expression by microarray using an Affymetrix human 133 plus 2.0 chip, and analyzed on Partek software. Pathway analysis of these carboplatin resistant cells was preformed using Ingenuity. Results: Virtually all melanoma tumor samples stained positively for CD271 by IHC, with a median of 50% cells within tumor samples expressing the cytoplasmic marker. Flow cytometry demonstrated an increase in the percentage of cells expressing CD271 as resistance to carboplatin increased. Wild type A375 cells contained 30% positive CD271, A375 6mg/mL resistant contained 42% positive and A375 10mg/mL resistant showed 66% positive. Microarray also exhibited a direct correlation between CD271 gene expression with increasing carboplatin resistance. Functional ontology enrichment revealed development and regulation of the EMT pathway as an important process impacted by our resistant cell line. Conclusions: The data suggests that as melanoma cells become resistant to certain chemotherapy drugs they essentially loose their differentiated phenotype and become more stem cell-like. Monitoring melanoma expression of CD271 could help individualize therapy and anticipate chemotherapeutic resistance in this high-risk group of patients.

7605 Background: In the setting of autologous stem cell transplantation (ASCT) in multiple myeloma (MM), it has been shown that peripheral blood absolute lymphocyte count (ALC) on day 15 is an independent prognostic factor for clinical outcomes. Recently the International Staging System (ISS) for MM has been developed as a simple staging system to assess survival in newly diagnosed MM patients. The role of ALC on survival in newly diagnosed MM patients is unknown. Methods: Between 1994 and 2002, 1,835 consecutive MM patients were evaluated at the Mayo Clinic, Rochester. Of these patients, we retrospectively analyzed 584 MM patients that were originally diagnosed and followed at the Mayo Clinic. The primary end point was to assess the role of ALC at the time of MM diagnosis on overall survival (OS). OS was measured from the date of diagnosis to time of death or last follow-up. ALC was analyzed as a continuous variable and dichotomized based on finding the optimal cut point based on the log-rank statistic. ALC was then compared to the ISS. Results: The median age of the cohort was 67 years (range: 29–94 years), including 234 females and 350 males. The median follow-up was 32 months (range: 1–136 months). The median ALC at diagnosis was 1.2 x 109/L (range: 0.12–5.44 x 109/L). ALC, as a continuos variables was identified as a prognostic factor for OS (HR= 0.466, 95%CI= 0.396–0.547, p < 0.0001). MM patients with an ALC ≥ 1.3 x 109/L experienced a superior OS compared with MM patients with an ALC < 1.3 x 109/L (55.5 months versus 22.6 months, p< 0.0001). In the multivariate analysis, ALC was independent prognostic factor when compared to the ISS (HR = 0.580, 95%CI=0.518–0.647, p< 0.0001). Conclusions: Our study shows that ALC at diagnosis for MM is an independent prognostic factor for OS, suggesting how the host immune status plays a critical role in the survival of patients with MM. No significant financial relationships to disclose.

7586 Background: The immunologic mechanisms of action of rituximab have been described as complement mediated lysis, vaccine like effect, antibody-dependent cellular cytotoxicity (ADCC) and the cellular microenvironment. We hypothesized that in the treatment of follicular grade 1 lymphoma (FL), the presence of a stronger host immune status prior to rituximab therapy would result in a prolonged time to progression (TTP). As a surrogate marker for immune status, we evaluated the absolute lymphocyte count (ALC) prior to rituximab treatment. Methods: Between 1996 and 2002, 1,104 consecutive FL patients were evaluated at Mayo Clinic Rochester. Of these patients, we retrospectively analyzed a group of all FL patients who received rituximab (375 mg/m2 once a week for four weeks) alone at any time during their lymphoma treatment at the Mayo Clinic (n=79). The primary end-point was to assess the impact of ALC just prior to rituximab therapy on TTP for FL. Results: The median age of the cohort was 56.6 years (range: 25–98 years). The median follow-up was 12.5 months (range: 1–76 months). An ALC count of ≥ 890 cells/μL prior to rituximab therapy predicted a longer TTP compared with an ALC < 890 cells/μl (25 months versus 8 months, respectively, p < 0.0124). A higher complete response rate was observed in the ALC ≥ 890 cells/μL group compared with the ALC < 890 cells/μL group [15/40 (38%) vs 5/39 (13%), p < 0.035]. The groups were balanced regarding the Follicular Lymphoma International Prognostic Index (FLIPI) (p = 0.794). Multivariate analysis demonstrated ALC ≥ 890 cells/μL prior to rituximab therapy as an independent prognostic factor for TTP when compared to hemoglobin, LDH, and Ann Arbor stage. The ALC was independent of the FLIPI in multivariate analysis. Conclusions: This data supports the hypothesis that a higher lymphocyte count, as a marker of the immune status of the patient, predicts for a longer TTP following rituximab therapy. No significant financial relationships to disclose.

19002 Background: Preliminary data suggesting positive clinical outcome in patients treated with adjuvant granulocyte macrophage colony stimulating factor (GM-CSF) following surgical resection of stage III/IV melanoma already exist (Spitler et al). A compassionate use protocol for the treatment of such patients with the “Spitler” regimen (GM-CSF administered at 250 mcg/day s.c. for 14 days of a 28 day cycle) has been ongoing at Mayo Clinic. Herein we present a retrospective review. Methods: Between 1998 and 2006, data was collected from 30 patients rendered disease free by surgery who received adjuvant GM-CSF. GM-CSF was administered per the “Spitler” regimen, although one patient received a reduced dose of 125mcg/day. The Kaplan-Meier approach was used to estimate relapse free survival. Results: Thirty patients (14 Female) with a median age of 53 and excellent performance status were evaluated. Most patients had resected stage III melanoma (Stage II/III/IV: 1/20/9). Median followup was 33 months (3–96). Twenty one patients were treated with GM-CSF for at least 12 months. Overall, relapse-free survival at 1 year was estimated to be 69% (95% confidence interval (CI) of 54% to 88%), reducing to 44% (95% CI of 29% to 67%) at 3 years. According to stage, relapse free survival at 1 year was 70% (III) and 63% (IV), and at 3 years was 54% (III), and 13% (IV). Among the 21 patients completing at least 1 year of therapy, relapse free survival at 3 years was 52%. Seventeen patients relapsed (III/IV: 10/7). Six of these patients were retreated and only two relapsed. Toxicities were grade 1 with most common being injection site rash (23%), asthenia (17%), myalgia (17%), and fever (7%). One patient required dose modification for elevated liver function tests. Conclusions: Although limited in scope, our data further support a potential beneficial effect of adjuvant GM-CSF in resected stage III/IV melanoma. Recurrences in the treated population were often localized and amenable to further surgical treatment. The clinical relevance of this strategy is currently being prospectively tested (E4697, now closed to accrual). No significant financial relationships to disclose.

18505 Background: Besides radiation, the immunologic mechanisms of action of 90Y ibritumomab tiuxetan (Zevalin) radioimmunotherapy (RIT) have been attributed to complement mediated lysis and antibody-dependent cellular cytotoxicity (ADCC). We hypothesized that a stronger host immune system prior to Zevalin therapy for relapsed follicular (grade 1&2) lymphomas (FL) would result in an improved time to progression (TTP). As a surrogate marker of host immune status, we studied absolute lymphocyte count (ALC) prior to Zevalin therapy and its impact on TTP in relapsed FL patients. Methods: Between 1996 and 2006, 75 patients with relapsed FL were treated with single agent Zevalin (0.4 mCi/kg, maximum of 32 mCi) at the Mayo Clinic. ALC was obtained from the complete blood cell count prior to RIT. Results: The median age of the cohort was 60 years (range, 29–82 years). 36% (27/75) patients were rituximab refractory. The median TTP in all patients was 6.4 months (range, 1–99+ months). Univariately, ALC as a continuous (HR = 0.448, p < 0.006) or dichotomized [ALC = 1.0 x 109/l (HR = 0.684, p < 0.007)] variable was identified as a prognostic factor for TTP. Superior TTP was observed with an ALC = 1.0 x 109/l (N = 32) compared with an ALC < 1.0 x 109/l (N=43) (median: 12.4 months vs 6.5 months, respectively, p < 0.007). Both groups were balanced in regard to the Follicular Lymphoma International Prognostic Index (FLIPI). ALC, as a continuous (HR = 0.507, p < 0.02) or dichotomized [ALC = 1.0 x 109/l (HR = 0.697, p < 0.01)] variable was identified as an independent prognostic factor for TTP in the multivariate analysis when compared with FLIPI. In the ALC = 1.0 x 109/l group, 14/32 (44%) of FL patients achieved a TTP = 12 months compared to only 7/43 (16%) in the ALC < 1.0 x 109/l group, (p < 0.02). Conclusions: This study supports our hypothesis that a higher ALC, as a marker of immune status of the patient, predicts longer TTP following Zevalin therapy in relapsed FL. No significant financial relationships to disclose.